Dietary calcium supplementation enhances efficacy but also toxicity of EGFR inhibitor therapy for colon cancer

Abstract

The inverse correlation between levels of dietary calcium and colorectal cancer (CRC) incidence has been extensively investigated. However, the impact of supplemental calcium on cancer therapy remains unknown. We used four models of CRC, Caco-2 and HCT116 human cancer cell lines and Apc^Min/+ and azoxymethane carcinogen-induced mouse models, to investigate the impact of a Western-style diet low in calcium (0.05%) vs. a similar diet but supplemented with calcium (5%) on therapeutic targeting of the epidermal growth factor receptor (EGFR). We found that calcium supplementation combined with pharmacologic blockade of EGFR results in an additive effect on tumor growth inhibition in all models. Unexpectedly, the combined use of dietary calcium supplementation and EGFR inhibitors also resulted in elevated toxicity suggesting that careful consideration be given when combining dietary supplements with prescribed cancer therapies.

Keywords:

• colon cancer
• EGFR
• calcium
• mouse models
• AG1478
• therapy
• toxicity

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Author Contributions

E.S.R. and D.W.T. designed all experiments, and wrote edited the manuscript. E.S.R. and E.B. performed all experiments.

Acknowledgments

Support was provided by National Institutes of Health grants CA105417 and CA092479 (DWT) and fellowship AT002835 (ESR). Infrastructure was supported by center grants CA016086 and DK034987. We would like to extend our gratitude to Dr. Ethan Lange of the Department of Genetics at UNC for consultations regarding the statistical analysis for this study.

Figures and Tables

Figure 1 Experimental design. At weaning (Apc^Min/+) or 1 week following the last carcinogen treatment (AOM) mice were randomly assigned to a control (0.05% calcium) or calcium (5% calcium) supplemented diet, with or without small-molecule EGFR inhibitor AG1478. Apc^Min/+ mice were treated for nine weeks and body weight and food consumption was measured at the beginning and end of treatment. AOM mice were treated for four months and body weight and food were monitored biweekly throughout the treatment period.

Figure 2 Effect of calcium on AG1478-mediated tumor (A) number and (B) size in Apc^Min/+ and (C) tumor number in AOM models. Values are expressed as a percent of control treatment. Number of mice (A and C) and number of tumors (B) are displayed in bars of graph. Equal numbers of males and females were used and data for both sexes are shown. (C) A significant difference in tumor number is observed in AG1478 compared with Calcium/AG1478 treated mice only in females, but data for both sexes is shown. *p < 0.001; **p < 0.0001. Error bars show SEM.

Figure 3 MTT assays of Caco-2 and HCT116 cell viability treated with AG1478 and/or calcium. (A) Calcium chloride and (B) AG1478 decrease cell viability compared with the control (no treatment) in a dose dependent manner. Supplementing a constant dose of AG1478 with increasing doses of calcium chloride results in reduced cell viability in an additive manner in both (C) Caco-2 and (D) HCT116 cells. Numbers within the bars indicate percent reduction when compared with control untreated cells. Error bars show SD.

Figure 4 Effect on signaling after AG1478 and calcium treatment. (A) In vivo: mice maintained on a control or calcium supplemented diet, with or without AG1478, were injected with phosphatase inhibitor prior to euthanasia. EGFR activation was measured in liver protein lysates from Apc^Min/+ and AOM mice, separately for each strain and sex. (B) In vitro: protein was extracted from human CRC cells lines after a 4 h treatment with or without 4 µM (Caco-2) or 8 µM AG1478 (HCT116) with 3.5 mM (“+”) and 4.5 mM (“++”) calcium chloride. (C) pEGFR ELISA was performed using protein extracted from cells lines after 2 h treatment with or without 4 µM (Caco-2) or 8 µM AG1478 (HCT116) with 3.5 mM (“+”) and 4.5 mM (“++”) calcium chloride followed by 20 min treatment with EGF.

Figure 5 General health of mice. (A) Comparison of body weight change for Apc^Min/+ and AOM mice maintained on a control (solid lines) or calcium (dashed lines) supplemented diets, with or without AG1478. Blood collected at the end of treatment was used for measurement of (B) serum calcium and (C) alanine aminotransferase (ALT) levels. The normal lower and upper levels are indicated for each test. Error bars show SEM.